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When it comes to slowing Aβ production as a therapeutic strategy for Alzheimer disease, blocking either β- or γ-secretase has proven effective but comes with nasty side effects. In each case, toning down the inhibition lessens the untoward consequences but compromises efficacy. A study in AD mice now suggests that a controlled one-two punch—hitting both enzymes at once, but moderately—might be the ticket to balancing efficacy and safety. Philip Wong of Johns Hopkins University, Baltimore, Maryland, presented some of this data at the 2008 Eibsee Meeting on Cellular Mechanisms of Alzheimer Disease in Germany (see ARF related news story). The full findings were published online in the January 6 issue of Science Translational Medicine.

As the two enzymes responsible for cleaving Aβ peptide out of its parent amyloid precursor protein (APP), β-secretase (BACE1) and γ-secretase have long captured the attention of AD drug developers. The problem is that these proteases cut things other than APP, and inhibiting them not only curtails Aβ production but also shuts down other critical biological processes. Wong and others have reported that BACE1 knockout mice have hypomyelinated axons (Willem et al., 2006), impaired synaptic function and cognition (Laird et al., 2005), as well as schizophrenia-like behaviors (Savonenko et al., 2008). Blocking γ-secretase activity in mice throws off Notch and epidermal growth factor signaling, causing skin tumors and gastrointestinal side effects (Li et al., 2007). Wong’s lab showed that reducing BACE1 or γ-secretase activity by just 30 to 50 percent was enough to prevent most of the unwanted side effects—however, with dampened CNS benefits. “We thought, Could we increase the efficacy in terms of reducing amyloidosis while limiting the side effects?” Wong said. “Since we knew both enzymes are required for generation of Aβ, perhaps they work in an additive manner.”

First author Vivian Chow and colleagues put this question to the test by creating three new lines of APP/PS1 mice: one lacking an allele of BACE1, one lacking an allele of Aph-1a (an essential component of the γ-secretase complex), and one heterozygous for both BACE1 and Aph-1a. Measuring Aβ load using biochemical and immunohistochemical analyses, the researchers found far less amyloid in the brains of BACE1+/- Aph-1a+/- APP/PS1 mice than in either of the single heterozygote APP/PS1 mice. This relative trend held up even as the animals aged to 19-23 months. APP/PS1 mice normally show advanced stages of Aβ pathogenesis by that age. Furthermore, amyloid reduction seemed to correlate with functional benefits. Compared with the APP/PS1 single heterozygotes, BACE1+/- Aph-1a+/- AD mice were better protected from memory deficits, to an extent that their performance in a Morris water maze was comparable to that of wild-type mice. Importantly, the double heterozygote APP/PS1 mice had a normal lifespan and seemed free of the other untoward side effects seen in animals completely lacking either secretase.

For the most part, the above outcomes were expected, Wong told ARF. However, “we were a little bit surprised that we got such a dramatic effect functionally,” he said. Consistent with them being key mediators of synaptic dysfunction, the researchers found reduced amounts of oligomeric Aβ in brain tissue from BACE1+/- Aph-1a+/- AD mice, whereas brain levels of Aβ oligomers hardly changed in single heterozygote AD mice. “That might be one reason why we got such a robust recovery of learning and memory in the double reduced mouse models,” Wong said.

Whatever the reason, the fact that modest reduction of both secretases could curb amyloid accumulation and preserve cognition, while avoiding harmful side effects in AD mice, suggests that combination strategies may eventually hold promise in the clinic. It remains to be seen whether the benefits of secretase-targeting compounds offered to AD patients in mid- to late life mirror the effects of genetic deletions present from birth in mice. In the meantime, the current study provides an important framework for evaluating future experimental drugs. “Genetic models define the limits of the mechanism-based toxicities,” Wong said. If compounds carry other toxicities not seen in the animal models, that would suggest the effects are due to the compound itself and may not relate to inhibition of APP processing.

The combination therapy could have benefits above and beyond blocking Aβ production. In a PNAS paper published in December, Ralph Nixon and colleagues at the Nathan Kline Institute in Orangeburg, New York, showed that another APP fragment—C-terminal product of β-secretase cleavage (βCTF)—is toxic early in AD (see ARF related news story). If true, this would mean that manhandling Aβ isn’t enough, offering further support for a multipronged attack on APP processing. As Wong noted, Nixon’s study suggests that “lowering the activity of γ-secretase alone may not be sufficient to deal with both problems of Aβ and βCTF accumulation.” (See full comment.)

Nixon agrees. “I think the stories are very compatible,” he said of his and Wong’s studies in an e-mail to ARF. “The partial inhibition of both secretases is quite interesting and could easily be viewed as implicating βCTF as well as Aβ. In addition to the mechanism Wong proposes of additive/synergistic effects on Aβ lowering, our data would suggest that an additional explanation for the combined inhibitor effects on memory deficits is a reversal of separate adverse effects of βCTF and Aβ on cognitive performance.” (See full comment below)

In the meantime, understanding why BACE1 inhibitors can be so toxic would speed their clinical development, which currently lags behind that of γ-secretase inhibitors. Teasing out BACE’s physiological roles would surely help. Toward this end, a team led by Dennis Selkoe, Brigham and Women’s Hospital, Boston, has used quantitative proteomics to identify 63 potential new substrates for BACE1. First author Matthew Hemming and colleagues probed two BACE1-overexpressing epithelial cell lines (HEK and HeLa) for proteins whose levels rose in parallel with increased BACE1 activity. More than half of the proteins are involved in intercellular communication or are receptors with recognized biological functions. If these are real substrates, then they may highlight additional concerns about strongly inhibiting BACE1, Wong noted. However, since the studies were done using cell lines that express artificially high levels of BACE1, the physiological relevance of the identified proteins remains in question, he said.

Other scientists did not see the BACE1 overexpression in these studies as a caveat as much as a possible means for discovering roles for β-secretase in conditions where its levels go up, such as traumatic brain injury and energy deprivation. The study “is particularly interesting because it demonstrates that increased β-secretase activity results in alteration of normal cellular functions, e.g., missorting of lysosomal hydrolases and increased cleavage of cell adhesion molecules, which may be detrimental for the cell,” wrote Giuseppina Tesco, Tufts University, Boston, in an e-mail to ARF. (See full comment below.) “Given that BACE1 is elevated in AD brains, increased cleavage of additional substrates may contribute to AD pathology. Thus, β-secretase inhibition is expected to be beneficial in conditions associated with increased levels of BACE1.”—Esther Landhuis

Comments on News and Primary Papers

This is very interesting work. The identification of over 60 BACE1 substrates using an unbiased and quantitative proteomic method sheds light on the role of β-secretase in conditions where BACE1 levels are increased. Levels of BACE1 are normally very low in peripheral tissues as well as in the epithelial cell lines used in this study. Moreover, cerebral BACE1 levels decrease postnatally (Willem et al. 2006). Altogether these findings indicate that in normal conditions, β-secretase activity is low. Instead, several groups, including ours, have reported that β-secretase activity is increased in stress conditions (cerebral ischemia, traumatic brain injury, energy deprivation, hypoxia, apoptosis). Thus, this study is particularly interesting because it demonstrates that increased β-secretase activity results in alteration of normal cellular functions, e.g., the missorting of lysosomal hydrolases and increased cleavage of cell adhesion molecules, which may be detrimental for the cell. Given that BACE1 is elevated in AD brains, increased cleavage of additional substrates may contribute to AD pathology. Thus, β-secretase inhibition is expected to be beneficial in conditions associated with increased levels of BACE1.

Ralph Nixon New York University School of Medicine/Nathan Kline Institute

Posted: 08 Jan 2010

The partial inhibition of both secretases is quite interesting, and the results could easily be viewed as implicating βCTF as well as Aβ in pathology. In addition to the mechanism Chow et al. propose of additive/synergistic effects on Aβ lowering, our data would suggest that an additional explanation for the combined inhibitor effects on memory deficits are a reversal of separate adverse effects of βCTF and Aβ on cognitive performance. A third copy of APP is known to be responsible for certain of the memory and learning deficits in the Ts65Dn mouse model of Down syndrome, which is associated with increased β-cleavage of APP but no measurable Aβ elevation, at least at the whole brain level (Choi et al. 2009; Salehi et al. 2006). In light of our recent evidence linking increased βCTF to AD-related endosome defects in Down syndrome and to FAD due to APP duplication, it is reasonable to suspect that βCTF may play a role in the rescue of cognitive deficits by BACE inhibition. This would be expected to add to effects of lowering Aβ levels in the APPswe/PS1DE9 model used in this study, which exhibits marked amyloid deposition/Aβ elevation in contrast to the Ts65Dn mouse.

Based on this framework, the caveats that our work suggested regarding the possible buildup of potentially toxic βCTF after using γ inhibitor alone would be offset by simultaneously blocking CTF buildup with a BACE inhibitor.

This is an interesting approach and could hold promise in the clinic. However, the paper describes a genetic knockdown approach. The progression of a small molecule combination therapy to the clinic would be more challenging.

Issues that would need to be addressed include assessing potential drug-drug interactions in humans. In the meantime, both tool γ-secretase and BACE inhibitors are available to test this theory in APP transgenic mice. These studies would need to be done to assess the potential of small molecule combinations and to assess any potential toxicity issues.

The β-secretase (BACE1) protease is one of the enzymes that cleaves the amyloid precursor protein (APP) and leads to the production of Aβ peptides. In Alzheimer disease, elevated expression of this enzyme is thought to underlie increased Aβ production and lead to pathological effects. As such, BACE1 is an attractive therapeutic target. However, as pointed out by the authors of this paper, the normal function of BACE1 is still not well understood. Although it was known that APP is not the only target of BACE1, few targets had been identified to date. In addition, in BACE1 knockout mice, various problems have been reported, suggesting that BACE1 has important roles in the nervous system. In this paper, the Selkoe laboratory performed a quantitative proteomic analysis to identify BACE1 substrates using cell culture systems in which they overexpress the protease. They identified 68 putative substrates and validated several of these. In addition, they identified various other proteins that are not membrane bound and are likely elevated by indirect effects. The putative substrates were identified in two cell lines that express very low levels of BACE1, and the results identified many differences between cells. This study illustrates the complexities associated with targeting the expression of BACE1 as a therapeutic strategy in Alzheimer disease and provides important fundamental information. It seems that the challenges moving forward will be to identify relevant substrates in neurons that express BACE1 and to determine the relative importance of various BACE1 substrates.

The new study by Chow and colleagues describes evidence that the combination of partial reductions in β-secretase and γ-secretase are effective in lowering Aβ production and deposition and preventing cognitive deficits in mice. This happens without the deleterious effects of complete knockout of either protease alone, something that many of us in the field have suspected for some time, but which had not yet been demonstrated.

The strategy for dual lowering of the two secretases was strictly genetic, not pharmacological: mice that are heterozygous knockouts of BACE1 and Aph1a were crossed into transgenic mice overexpressing AD mutant APP and Presenilin.

Nevertheless, the results provide an important proof of principle. From a practical perspective though, it will be difficult to test this combination approach in humans, as each secretase inhibitor must first be tested individually and shown to be efficacious before combination regimens can be tried.